The present invention relates generally to metal binding conjugates of antibodies and antibody fragments which are useful in a variety of diagnostic and therapeutic techniques. Specifically, the invention relates to methods for the preparation of improved aqueous solutions of chelator/antibody conjugates and metal ion/chelator/antibody conjugates.
In recent years significant progress has been made in efforts to produce pharmaceutical agents comprising conjugates of radioisotopic and other metal ions with monoclonal antibodies. The ability of monoclonal antibodies to bind with high specificity to selected antigenic epitopes provides a means by which specified metal ions may be selectively directed to target tissues. Conjugates of radioisotopic metal ions with monoclonal antibodies capable of selective binding with tumor and other disease associated antigens are contemplated to be of particular utility for use in diagnostic imaging and radiotherapy protocols for the diagnosis and treatment of conditions such as cancer.
Where antibody metal ion conjugates are to be used for the therapeutic delivery of cytotoxic radiation the metal is generally selected from a beta particle emitter such as yttrium. Order, et al., Int J. Radiation Oncology Biol. Phys., 12 277-81 (1986) describes treatment of hepatocellular cancer with antiferritin polyclonal antibodies to which .sup.90 yttrium has been chelated. Buchsbaum, et al., Int. J. Nucl. Med. Biol., Vol. 12. No. 2, pp. 79-82, 1985 discloses radiolabelling of monoclonal antibodies to CEA with .sup.88 yttrium and suggests the possibility of localization and treatment of colorectal cancers therewith. Nicolotti, EPO Applicatiom No. 174,853 published Mar. 19, 1986, discloses conjugates comprising metal ions and antibody fragments. According to that disclosure, monoclonal antibodies of subclass IgG are enzymatically treated to remove the Fc fragment and reductively cleave the disulfide bond linking the antibody heavy chains. The Fab' fragment is then linked to a chelating agent bound to a radionuclide metal ion for in vivo diagnostic or therapeutic use.
Where antibody conjugates are to be used for diagnostic radioimaging (radioimmunoscintigraphy) purposes a gamma emitting radioisotope is preferably selected. Goldenberg, et al., N. Eng. J. Med., 298, 1384-88 (1978) discloses diagnostic imaging experiments wherein antibodies t the known tumor associated antigen carcinoembryonic antigen (CEA) are labelled with .sup.131 iodine and injected into patients with cancer.
After 48 hours, the patients are scanned with a gamma scintillation camera and tumors are localized by the gamma emission pattern. Gansow, et al., U.S. Pat. No. 4,454,106 discloses the use of monoclonal antibody/metal ion conjugates for in vivo radioimaging diagnostic methods.
U.S. Pat. No. 4,472,509, Gansow, et al., discloses the use of diethylenetriaminepentaacetic acid (DTPA) chelating agents for the binding of radiometals to monoclonal antibodies. The patent is particularly directed to a purification technique for the removal of non-bonded and adventitiously bonded (non-chelated) metal from radiopharmaceuticals but is illustrative of art recognized protocols for preparation of radioisotopic pharmaceuticals. According to such general procedures, an antibody specifically reactive with the target tissue associated antigen is reacted with a quantity of a selected bifunctional chelating agent having protein binding and metal binding functionalities to produce a chelator/antibody conjugate. In conjugating the antibodies with the chelators an excess of chelating agent is reacted with the antibodies, the specific ratio being dependent upon the nature of the reagents and the desired number of chelating agents per antibody. Gansow, et al., disclose chelator to antibody ratios of 100:1 to 600:1 as being particularly useful according to one system for achieving from about 0.5 to 1.5 bound chelators per molecule. The reference cautions, however, that care must be taken not to add so many chelators per antibody molecule so as to adversely affect the immunoreactivity of the antibody. After conjugation of the chelators with the antibodies, the reaction mixture is purified to remove excess decomposed chelator. The Gansow reference discloses purification by dialysis of the conjugate mixture over a 48 hour period against three changes of solution comprising 50 mM citrate and 200 mM sodium chloride with 1 ml of gel resin. The reference also discloses an optional first dialysis step which may be carried out against a dilute solution of ascorbic acid (30 mM) and ethylenediaminetetraacetic acid (EDTA) chelating agent (5 mM) "to remove an residual iron which may be present in the chelate or the protein."
The purified chelator/antibody conjugate may then be conjugated with the active metal label or stored until later use. A solution of metal label is obtained from a source such as a radioisotope generator or accelerator according to known methods. Metal chelation is then carried out in an aqueous solution with pHs generally ranging from about 3.2 to about 9 so as not to impair the biological activity or specificity of the antibodies. Weakly chelating acids and bases such as citric acid and glycine are employed as buffers. The metal ions are generally introduced in the form of metal salts such as metal halides, nitrates or perchlorates with chlorides being particularly preferred. The reference suggests that the metal salt be employed in as high a concentration as is practicable although it notes that when radioactive metals are used, health and safety considerations recommend use of a metal concentration below one equivalent of metal per chelate binding site.
Gansow, et al., state that metal ion/chelator/antibody conjugates so prepared will generally require purification prior to their administration in vivo in order to remove free and adventitiously bonded metal. The patent discloses various techniques involving a combination of ion exchange and gel filtration chromatography. A preferred procedure involves passing an aqueous solution containing the conjugate through a chromatography column with two layers, a first layer selected from a group consisting of an anion exchange resin, a cation exchange resin and a chelating ion exchange resin and a second layer comprising a sizing matrix. Such a procedure is said to result in solutions which showed less than a six percent (6%) loss of indium when dialyzed against a buffer comprising 20 mM 2 (N morpholino)ethane sulfonic acid) (MES) and 200 mM sodium chloride at pH 6.0.
Numerous variations on the general methods disclosed above are known for the covalent labelling of antibodies and antibody fragments with metal chelating moieties. Such methods include those whereby the labelling proceeds via a mixed anhydride as disclosed by Krejcarek, et al., Biochem. Biophys. Res. Commun., 77, 581 (1977), via a bicyclic anhydride as described by Hnatowich, et al., J. Immunol. Methods, 65, 147 (1983) or via an active ester as described by Paxton, et al., Cancer Res., 45, 5694 (1985).
Such metal chelate labeled antibodies have been administered to patients in a variety of studies and certain properties have become recognized in the art as characteristic of the behavior of these conjugates in humans. The most common observation has been that metal chelate labeled antibodies accumulate in the liver to a greater extent than do antibodies labeled with a non metallic isotope such as iodine 131 (Larson et al, Nucl. Med. Biol., 15, 231 (1988)). This phenomenon constitutes the most important limitation on the clinical utility of metal labeled antibodies, as it frequently prevents the detection of tumor deposits in the liver, which is a major site of metastatic spread in many types of cancer (Larson et al, Nucl. Med. Biol., 15, 231 (1988); Beatty et al, Cancer Res., 46, 6494 (1986); Beatty et al, J. Surg. Onc., 36, 98 (1987); Abdel-Nabi et al, Radiology, 164, 617 (1987)). Other characteristics relate to the pharmacokinetic properties of metal chelate labeled antibodies. A common observation has been that antibodies exhibit biphasic serum clearance kinetics, with relatively rapid disappearance of a fraction of the injected dose of radioactivity (termed the .alpha.-phase) followed by a much slower clearance of the remaining activity (called the .beta.-phase) (Hnatowich et al, J. Nucl. Med., 26, 849 (1985); Murray et al Cancer Res., 48, 4417 (1988); Murray et al., J. Nucl. Med., 28, 25 (1987)). The volume of distribution of the injected radioactivity often exceeds the plasma volume of the patient and both serum clearance (t.sub.178 ) and the volume of distribution are often dependent on the dose of antibody administered. (Murray et al, Cancer Res., 47, 4417 (1988); Carrasquillo et al. J. Nucl. Med., 29, 39 (1988)). The sensitivity of detection of tumor sites has similarly shown a dose dependence (Carrasquillo et al, J. Nucl. Med., 29 39 (1988); Abdel Nabi et al, Radiology, 164, 617 (1987)). These data indicate that the injected labeled immunoglobulin does not remain solely in the plasma compartment, in which case monophasic serum kinetics and a volume of distribution that approximates the plasma volume would be expected, but rather distributes into additional compartments representing non-specific accumulation of antibody in non target organs such as liver. These compartments are in some cases saturable, and hence the observed dose dependence. Multi-compartmental kinetic models of antibody biodistribution in humans have been constructed (Egan et al, Cancer Res., 47, 3328 (1987)).
The methods of Gansow, et al., as are those of other workers in the field, are controlled by several purity related concerns relating to the preparation of metal ion/chelating agent/antibody conjugates for in vivo administration. It is axiomatic that the quantity of active metal (as distinguished from other metal impurities present in the conjugate solution in either free, adventitiously bound or chelated form) delivered to target tissues be maximized. It is similarly the case that the quantity of active metal delivered to non-target tissues be minimized. In diagnostic imaging protocols this is the case as a consequence of the desire to minimize background signals. In cytotoxic radiotherapy protocols, this is a consequence of concerns relating to cytotoxic effects on non target tissues. An additional concern relates to the desire that the quantity of antibody conjugates be minimized in these protocols in order to minimize the antigenic effects of in vivo introduction of protein.
The goal of directing a maximum quantity of active metal to the target tissue is affected by a number of factors. Foremost of these factors are the specificity and activity of the antibody. The antibody or antibody fragment upon which the conjugate is based must have high binding activity and selectivity for the target antigen. In addition, the antigen to which the antibody is specifically targeted must be selected such that the antigen has a high specificity for the tissue to be targeted as opposed to other tissues.
Given an antigen that is highly specific for the target tissue and an antibody with high activity and selectivity for that antigen, it is then a significant concern that the immunoreactivity of the antibody making up the conjugate not be diminished by virtue of some chemical alteration occurring during the process of forming the chelator/antibody conjugate or in the metal chelating step. Chemical alteration resulting in partial or total loss of immunological activity may occur as a consequence of high temperature, extremely acidic or alkaline pH or other chemical treatment. Such alteration may, in the extreme, result in denaturation of the antibody molecule. Alteration less extreme than denaturation may occur where the protein binding groups of bifunctional chelating agent react and link with amino acid residues or with glycosylation on the antibody molecules in such a manner that the specific binding regions of the antibody are altered or sterically blocked. The higher the chelator to antibody substitution level in any given chelator/antibody system, the greater the likelihood that such loss of immunoreactivity will occur.
A corollary to the desire to maximize the delivery of the active metal to the target tissue is the desire to minimize the delivery of the active metal to nontarget tissue. It is the case that active metal which goes to target tissues is almost invariably bound by means of a chelating agent to an antibody specific for that tissue. By contrast, the bulk of active metal which is delivered to nontarget tissues is free metal, metal which was covalently bound to an antibody which was not delivered to the target tissue or adventitiously bound metal which became free of the antibody in vivo. Active metal which is adventitiously bound or free in solution will frequently, upon administration to the patient, become rapidly bound to serum transferrin and be subsequently distributed primarily to the liver and bone marrow leading to undesirable nontarget accumulation of the radiometal. The presence of free metal in radioisotopic preparations for administration in vivo is of particular concern where the toxic effects of concentrating such metals at the liver and other nontarget organs stands to present major limitations to the use of such radioisotopic pharmaceuticals.
With respect to any preparation for administration in vivo it is particularly desired to minimize the quantity of antibodies utilized to deliver a quantity of active metal. This results from concerns related to the antigenicity of the antibodies and antibody fragments used to deliver active metals and the possibility of immune reactions to the pharmaceutical preparation itself. Efforts to minimize the quantity of antibodies introduced by increasing the quantity of metal ions bound to each antibody tend to be limited by the tendency of antibodies to denature or lose specific binding activity at elevated levels of chelating agent substitution.
A significant factor bearing on the various concerns recited above relates to the extremely high concentrations of impurities found in radiometal preparations available for conjugation with chelator/antibody conjugates. Of interest to the present invention is the disclosure of Hnatowich, et al., J. Immunol. Methods, 65, 147 (1983) which states that an antibody/chelator composition with a 1:1 chelator to antibody ratio and labelled with indium-111 (25 mCi/mg) has only 4% of the available chelating sites occupied by indium-111 atoms. The metal impurities present at elevated levels in available radiometal preparations effectively compete against the active label metals for binding sites on the chelator/antibody conjugates. The presence of such impurities mandates the use of chelator/antibody conjugates in quantities far greater than would be required to simply chelate a given amount of the pure metal label. The requirement of adding increased quantities of metal solution to assure chelation of a specified amount of active metal is undesirable because to do so results in the presence of greater quantities of free and adventitiously bonded metal which must generally be chelated or removed from the conjugate solution prior to administration in vivo. Use of greatly increased quantities of the chelator/antibody conjugates to chelate sufficient label is also undesirable because of the increased antigenicity of the administered dose.
Efforts to purify radiometal solutions prior to chelation with the conjugate solution have been disclosed as have been efforts to reduce the extent of prelabelling chelation of free chelator groups in the chelator/antibody conjugate. Meares, et al., Anal. Biochem. 142, 68 (1984) discloses the preparation of bifunctional EDTA analogues bearing isothiocyanate and bromoacetamide derivatives as substrate reactive groups. The chelator/antibody conjugates were labelled with indium-111 and other metal ions. The reference discloses the use of elaborate precautions to minimize metal contamination when performing the conjugate forming and coupling procedures. Such precautions include the use of high purity water, metal free buffer salts and acid-washed glassware and are useful for limiting the concentration of metal contaminants which compete for chelating sites with the desired metal. The reference also discloses the purification of commercially available .sup.111 InCl.sub.3 solutions by anion exchange chromatography to remove many of the contaminating metals present in the commercially available solution.
According to one procedure, Meares, et al., discloses preparation of the bromoacetamide analog of EDTA and its reaction with a mouse monoclonal anti transferrin receptor antibody solution at a 10:1 molar ratio. After incubation at 37.degree. C. for two hours, the reaction mixture was removed and applied to a Sephadex G-50-80 centrifuge column prepared with 0.1M ammonium citrate (pH 6) and a gel filtration step was carried out to remove unbound chelator. The purified chelator/antibody product had a concentration of 9.5.times.10.sup.-5 M which with a chelator/antibody ratio of 1.3:1 provided a chelator concentration of 1.24.times.10.sup.-4 M.
Carrier free .sup.111 indium stock solutions were prepared by adding to an ammonium citrate buffer solution an .sup.111 InCl.sub.3 solution which had been purified by treatment in a Bio Rad AGl-X4 anion exchange column equilibrated with 2M HCl. Two 10 ul aliquots of the .sup.111 indium solution were mixed with 5 ul aliquots of the EDTA/antibody solution and allowed to incubate for periods of time ranging from 5 to 80 minutes. An EDTA challenge procedure was carried out against samples of the indium/chelator/antibody conjugates in order to scavenge any free or adventitiously bound metal from the conjugate solution. One ul aliquots of the conjugate solutions were contacted with 5 ul aliquots of a 10 mM solution of Na2 EDTA challenge solution. The solutions so treated were then subjected to a thin layer chromatography (TLC) procedure by which antibody-bound metal was separated from free and chelator bound metal in order to determine the quantity of nonspecifically bound indium. The reference reported that the amount of unbound indium being transported on the TLC plate was only 3 to 5% of the total indium thus indicating that the radiochemical yield of the metal chelation procedure was 95 to 97%.
Meares, et al., state that in procedures where the metal ion is to be added last after preparation of the antibody/chelator conjugate that it is preferred that the concentration of protein bound chelating groups be greater than 10.sup.-5 M in order that the added metal ions may be bound quickly and quantitively. The reference states that with antibody concentrations in excess of 15 mg/ml (10.sup.-4 M) that the preferred conditions may be achieved.
Goodwin, et al., J. Nuc. Med., 26, 493-502 (1985) discloses further work of the Meares group wherein bromoacetamidobenzyl EDTA was conjugated to mouse monoclonal antibodies specific for the mouse major histocompatibility complex alloantigen IA.sup.k according the method of Meares, et al. described earlier. The antibodies are present at a concentration of 1.5.times.10.sup.-4 M which with a chelator to antibody ratio of 3.3:1 resulted in a 5.times.10.sup.-4 M concentration of chelators.
Radiolabelling was performed by combining small (10 to 50 ul) aliquots of the antibody/chelator conjugate with 50 ul aliquots of purified .sup.111 indium citrate at pH 5.0. Chelation of the indium to the chelator/antibody conjugate was said to go to completion in less than 5 minutes. The reference indicated that the indium label could not then be removed from the conjugate even with a greater than thousand fold challenge with EDTA. Labelling efficiency and radio chemical yield were measured by the EDTA challenge/thin layer chromatography procedure of Meares, et al. which indicated radiochemical yields ranging from 85 to 95%. In some experiments unbound metal was complexed with an EDTA chase. In other experiments, the indium/chelator/antibody solution containing unbound metal was diluted in phosphate buffered normal saline containing 0.1% human serum albumin, 0.1M sodium citrate and 0.01M EDTA which bound free metal but was not removed prior to intravenous injection. It is understood that the theory behind the addition of unconjugated chelators to the solution is that such agents will chelate with free and adventitiously bound metals and that the resulting chelate when administered in vivo will be rapidly cleared from the circulation by way of the kidney. The reference discloses animal biodistributionstudies with the indium/chelator/antibody conjugate. Organ distribution studies after 24 hours showed spleen uptakes in excess of about 100% dose per gram of organ for antigen positive mice although spleen uptakes were lower for antigen negative mice and in systems where the conjugate comprises a normal mouse IgG. The reference noted that indium chelates have increased stability over radioiodine preparations and that such stability produces not only increased target concentrations but also higher nonspecific blood and liver activity.
Of interest to the present invention is the disclosure of Zoghbi, et al., Invest. Radiol., 21 710 (1986) which relates to procedures for the purification of indium-111. The reference notes that there is large variability in the purity of commercially available indium and that variability in the quantities of indium chelated by chelators such as DTPA (well below the capacity of the ligands) may be explained by the presence of cationic contaminants such as zinc, iron and aluminum in commercial indium preparations and the fact that the chelators are not specific for indium. The reference reports that .sup.111 indium purified according to a solvent extraction procedure disclosed by the reference exhibits more than a threefold increase in the specific activity of an .sup.111 In-DTPA-monoclonal antibody over .sup.111 indium which was not purified.
Later references by the Gansow group disclose various post-chelation step purification procedures for the indium/chelator/conjugate solutions. Brechbiel, et al., Inorg. Chem., 25, 2772 (1986) and Esteban, et al., J. Nucl. Med., 28, 861 (1987), compare several different procedures for removing unbound indium-111 from the monoclonal antibody B72.3. Brechbiel, et al., compares (1) the EDTA chase method of Goodwin, et al., (2) gel column chromatography, (3 high performance liquid chromatography (HPLC) and (4) the combination of gel column chromatography and HPLC purification. The reference states that the simple addition of free EDTA to the mixture was not sufficient to remove all unreacted .sup.111 indium by localization and passage through the kidneys but that instead the metal tended to localize in the liver. The preferred method for purification of the conjugate solutions was stated to be passage through a Sephadex G-50 column followed by HPLC purification on a TSK 3000 sizing column. Treatment by HPLC was stated to be the only method for removal of "high molecular weight" aggregates such as cross linked chelating moieties. The reference recommends use of the strongest possible chelating agent in combination with mild coupling techniques and the best purification techniques.
Esteban, et al., J. Nucl. Med., 28, 861 (1987) provided description of additional purification and biodistribution studies of the Gansow group with the B72.3 monoclonal antibody. Chelator/antibody conjugates with bifunctional DTPA and EDTA were formed at various substitution ratios. The antibody chelate immunoreactivity was found to vary significantly with the molar ratio with the conjugates retaining 100 of their immunoreactivity when compared to unmodified IgG at 1:1 ratios. Chelator to antibody ratios of 3:1 or greater resulted in the loss of over 50% of immunoreactivity. Biodistribution studies were conducted with indium labelled conjugates having chelator to antibody ratios of 1:1. The reference confirmed the statement of the Brechbiel, et al., reference that the use of excess EDTA to chelate free metal in the conjugate solution provided poor purification with tumor to liver ratios of less than 1.6:1 while the use of labelled conjugates purified by HPLC resulted in tumor to liver ratios of 4.6:1.
Of interest to the present invention are references relating to efforts to systematically establish the maximum number of chelating groups which may be incorporated into each antibody molecule. Paik, et al., J. Nucl. Med., 24, 1158 (1983) and J. Nucl. Med., 24 932 (1983) disclose the optimization of, respectively, bicyclic DTPA anhydride coupling to monoclonal antibody 17-1A and mixed anhydride coupling of DTPA to a monoclonal antibody to human serum albumin. Generally, however, those skilled in the art have felt little need to push substitution levels beyond one chelating group per antibody molecule since indium-111 is readily available in carrier free form.
These various procedures involving prelabelling purification of the metal ion solution or post labelling chasing or purification of the metal ion/chelator/antibody conjugate solution are time consuming and difficult to perform under conditions which preserve the sterility and apyrogenicity essential to an injectable radiopharmaceutical for human use. Moreover, the setting in which such antibody radiolabelling procedures are to be routinely performed is typically that of a hospital nuclear pharmacy and such facilities frequently lack the equipment and trained personnel to carry out the most preferred yet most complex post-purification techniques such a HPLC and gel chromatography.